1. Field of the Invention
This invention relates generally to bifunctional chelating agents for use with biologically active peptides. More particularly, the present invention relates to the incorporation of ligands directly into peptides at nonbiologically active locations such that the ligand will bind the desired metal ion and the resulting complex will serve as a bifunctional agent and as a spacer molecule.
2. Technology Background
Scintigraphic imaging and similar radiographic techniques for visualizing tissues in vivo are finding ever-increasing application in biological and medical research and in diagnostic and therapeutic procedures. Generally, scintigraphic procedures involve the preparation of radioactive agents which upon introduction to a biological subject, become localized in the specific organ, tissue or skeletal structure of choice. When so localized, traces, plots or scintiphotos depicting the in vivo distribution of radiographic material can be made by various radiation detectors, e.g., traversing scanners and scintillation cameras. The distribution and corresponding relative intensity of the detected radioactive material not only indicates the space occupied by the targeted tissue, but also indicates a presence of receptors, antigens, aberrations, pathological conditions, and the like.
In general, depending on the type of radionuclide and the target organ or tissue of interest, the compositions comprise a radionuclide, a carrier agent such as a biologically active protein or peptide designed to target the specific organ or tissue site, various auxiliary agents which affix the radionuclide to the carrier such as bifunctional chelating agents, water or other delivery vehicles suitable for injection into, or aspiration by, the patient, such as physiological buffers, salts, and the like. The auxiliary agent attaches or complexes the radionuclide to the peptide carrier agent, which permits the radionuclide to localize where the carrier agent concentrates in the biological subject.
Often the biologically active protein or peptide has one or more bioactive sites separated by nonbiologically active peptide sequences. For example, Hirulog, a thrombin binding peptide, contains a spacer constructed between a catalytic binding site tripeptide and a 12-mer sequence necessary for anion binding excosite (ABE) site binding. In the Biogen hirudin analog, D-Phe-Pro-Arg-(Gly).sub.n -Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu, the polyglycine unit with n from 4 to 8 merely acts as a spacer. In thrombin binding assays, replacement of tetraglycine with hexa- and octaglycine units have very little effect on the binding constants, K.sub.i. J. M. Maraganore, P. Bourdon, J. Jabloski, K. L. Ramachandran, J. W. Fenton, Biochemistry, vol. 29, pp. 7095 (1990).
There are other examples of biologically active peptides having nonbiologically active spacers. For example, in Melanin Concentrating Hormone (MCH), 7-amino-hepatanoic acid has been used as a liner replacement of cystine. D. W. Brown et al., Biopolymers, vol. 29, p. 609 (1990). In addition, analogs of Atrial Natriuretic Factor (ANF) have been prepared using -HN(CH.sub.2 CH.sub.2 CH.sub.2).sub.3 CH.sub.2 CO- and -HN(CH.sub.2 CH.sub.2 O).sub.3 CH.sub.2 CO. These fragments replace tetraamino acid residues in the ANF. D. Boumrah, et al. Tetrahedron Letters, vol. 32, no. 52, p. 7735 (1991).
When using biologically active peptides which have nonbiologically active spacers, it is important that the bifunctional chelating agent not destroy the peptides's bioactivity. If the peptide's bioactivity is destroyed, then the peptide subsequently labeled with a radioisotope will have little value in diagnostic or therapeutic application.
It will be appreciated that it would be an advancement in the art to provide ligands capable of forming chelate complexes with diagnostic and therapeutic radioisotopes which can be incorporated directly into peptides at nonbiologically active locations such that the ligands function as spacer compounds.
Such ligands and methods of incorporating the ligands into peptides are disclosed and claimed herein.